Cancer is one of the major diseases that cause human clinical death, especially malignant tumors such as lung cancer, gastric cancer, breast cancer, pancreatic cancer, liver cancer, intestinal cancer, esophageal cancer and leukemia, which have extremely high mortality rates. However, until present, no effective methods and drugs are available to prevent, cure and eradicate cancer. Therefore, there is an urgent need for high-quality drugs and therapeutic methods against cancer with good specificity, high activity, low toxicity and no drug resistance.
Leukemia, also known as blood cancer, is a clonal malignant disease of hematopoietic stem cells. Since leukemia cells lose the ability to differentiate into mature functional blood cells and induce malignant proliferation by stagnating at different stages of hematopoietic cell development, they proliferate and accumulate in bone marrow and other hematopoietic tissues, and infiltrate other organs and tissues, causing the suppression of normal hematopoiesis. Its clinical manifestations include anemia, hemorrhaging, infection and infiltration of various organs, etc. The incidence of leukemia ranks 6th/7th among all tumors, with an especially high incidence in children and the elderly. Leukemia is a heterogeneous cell malignancy, with multiple varieties, a complicated pathogenesis and different clinical features. Some leukemias are characterized by rapid onset, high mortality, short survival time, a higher susceptibility for relapse, poor prognosis and difficult to cure. For example, the five-year survival rate of acute myeloid leukemia (AML) in patients over 60 years old is only 10% to 20%, while it is 40% to 50% in patients under 60 years old (Dohner H et al., Acute Myeloid Leukemia. N Engl J Med. 2015; 373 (12): 1136-52).
Although blood cancer can be treated by a variety of methods, such as chemotherapy, radiotherapy, immunotherapy, targeted therapy, induced differentiation therapy and bone marrow/stem cell transplantation, clinical therapies for the treatment of AML have undergone almost no changes in the past 40 years, and nowadays its standard treatment method is still based on remission induction, namely, “7+3” basic therapy (daunorubicin 25-45 mg/m2, IV days 1-3, cytarabine 100 mg/m2, IV days 1-7, except acute promyelocytic leukemia APL). Although classic chemotherapy regimens can effectively induce the remission of AML in a short period of time and suppress or kill cancer cells, these chemotherapeutic agents have adverse side effects, poor selectivity, and are easier to cause recurrence and drug resistance, resulting in AML cannot be completely cured with chemotherapy. Targeted therapy and tumor immunotherapy is the main development direction for the clinical treatment of cancer. Targeted therapy has good specificity, fewer side effects, and an obvious curative effect. Many targeted-therapeutic drugs have been successfully applied to different types of cancer including some types of leukemia, such as the Gleevec for CML treatment. However, no effective targeted-therapeutic drugs that are approved for the treatment of AML have been put on the world market so far. Therefore, there is an urgent need for a large number of drugs treating AML in clinical practice.
According to the cytogenetic genome big data analysis of clinical specimens from AML patients, the frequent occurrence of gene mutations is a major characteristic of AML. For example, the genetic mutation rate related to cell signaling pathways is about 50-60%, the rate of genetic abnormality related to DNA methylation is 44%, the rate of chromosomal modification gene mutations is approximately 30%, the rate of myeloid transcription factor gene abnormalities is 20%-25%, the occurrence rate of transcription factor fusion genes is approximately 18%, and the rate of tumor suppressor gene mutations is 14%. In AML patients, the most common genetic abnormalities include for example FLT3-ITD (19%-28%), FLT3-TKD (5%-10%), NPM1 (mutation rate of 27%-35%), DNMTA (mutation rate of 26%), NRAS (mutation rate of 8%-9%)), ASXL1 (mutation rate of 17%-19%), CEBPA (mutation rate of 4%-6%), TET2 (mutation rate of 8%-27%), WT1 (gene abnormality rate of 8%), IDH2 (point mutation rate of 8%-9%), IDH1 (mutation rate of 9%), KIT (mutation rate of 2%-4%), RUNX1 (mutation rate of 5-10%), MLL-PTD (5%), PHF6 (3%), KRAS (mutation rate of 2-4%), TP53 (mutation rate of 2%-8%), EZH2 (mutation rate of about 2%), JAK2 (mutation rate of 1%-3%) (Coombs C C et al., Molecular therapy for acute myeloid leukaemia. Nat Rev Clin Oncol. 2016; 13, 305-318. Welch J S et al., The origin and evolution of mutations in acute myeloid leukemia. Cell. 2012; 150:264-278. Kandoth C et al., Mutational landscape and significance across 12 major cancer types. Nature 2013; 502(7471):333-339. Ding L et al., Clonal evolution in relapsed acute myeloid leukaemia revealed by whole-genome sequencing. Nature. 2012; 481:506-510. Hanahan D et al., The hallmarks of cancer. Cell. 2000; 100: 57-70. The Cancer Genome Atlas Research Network Genomic and epigenomic landscapes of adult de novo acute myeloid leukemia. N. Engl. J. Med. 2013; 368:2059-2074).
Protein kinase is a kinase enzyme that phosphorylates proteins and is essential for important cellular physiological functions such as cell growth, development, differentiation, metabolism, aging, and apoptosis. There are two major types: transmembrane protein kinases and cytosolic protein kinases. Protein kinase abnormalities can directly lead to clinically distinct diseases such as cancer, inflammation, immune system and nervous system disorders, cardiovascular and cerebrovascular diseases. After decades of continuous efforts, many protein kinases such as EGFR, HER2/3/4, VEGFRs, PDGFRs, c-MET, IGF-1R, FGFRs, CSF-1R, TRK receptors, Ephrin receptors, TAM receptors, TIE-2, FLT-3, RET, ALK, BCR-ABL, JAKs, SRC, FAK, BTK, SYK, BLK, CDK, PI3K, MEK/RAS/RAF have been identified as target protein molecules for different diseases. Some of these protein kinase inhibitors have been successfully used in clinical applications as targeted therapies and shown good therapeutic effects, such as BCR-ABL, EGFR/HER2, ALK, BTK, VEGFR, JAK and other protein kinase inhibitors. FMS-like tyrosine kinase 3 (FLT3), also known as fetal liver kinase-2 (FLK-2) or human stem cell kinase-1 (STK-1) belongs to the type III receptor tyrosine kinase. The kinase family includes the colony stimulating factor 1 receptor (CSF1R), platelet-derived growth factor 1 receptor (PDGFR α/β) and stem cell factor receptor KIT. Under normal growth and development physiological conditions, expression of FLT3 gene mainly occurs in the early development of the brain, liver, placenta, gonads, and hematopoietic cells. During the growth and development of myeloid and lymphoid stem cells, the FLT3 gene and its ligand gene FLT3-L are highly expressed. When FLT3-L binds with FLT3, it induces FLT3 protein autophosphorylation and activates FLT3 enzyme activity and its mediated downstream signaling pathways such as PI3K, JAK/STAT, and RAS, and participates in biological functions such as blood cell growth, development, proliferation, and differentiation (Drexler H G et al., FLT3: receptor and ligand. Growth Factors. 2004; 22(2):71-3. Review). For example, in FLT3 gene deficient mice the number of myeloid and lymphoid progenitor cells is reduced. However, when the FLT3 gene is abnormally expressed or mutated, normal blood cells become canceration and leukemia develops. For example, about 30% of patients with acute myeloid leukemia (AML) exhibit mutations in FLT3 Internal Tandem Duplication (ITD, 19-28%) and tyrosine kinase domain mutation (TKD, 5%-10%). In patients with myelodysplastic syndrome (MDS) with moderate or severe risks, the mutation rate of FLT3 is 2%; in APL patients, the mutation rate is less than 5%; the incidence in ALL is less than 1%, which mainly occurs in cases with double phenotype ALL.
The two kinds of FLT3 mutation (FLT3-ITD and FLT3-TKD), including the FLT3-ITD/FLT3-TKD double mutation, can cause FLT3 protein autophosphorylation, resulting in FLT3 ligand-independent constitutive activation and abnormal downstream signal transduction, thereby promoting the malignant proliferation of leukemia cells and inhibiting normal cellular apoptosis. The FLT3 tyrosine kinase constitutively active mutation is one of the primary mutations in AML and one of the major causes of AML. Because FLT3-ITD clones have selective growth advantages, it is difficult to cure this type of leukemia using common chemotherapeutic drugs alone. Furthermore, patients with this type of leukemia have a higher tolerance for chemotherapeutic drugs and have poor clinical prognosis. Patients are prone to develop resistance to chemotherapeutic agents and relapse, therefore the FLT3 tyrosine kinase activating mutation has become an important target for AML targeted therapy (Gilliland D G, Griffin J D. The roles of FLT3 in hematopoiesis and leukemia. Blood. 2002; 100(5):1532-1542. Kiyoi H et al., Internal tandem duplication of the FLT3 gene is a novel modality of elongation mutation which causes constitutive activation of the product. Leukemia. 1998; 12(9): 1333-1337).
The field of AML research in regards to drug development has always been most concerned with FLT3-ITD and FLT3-TKD inhibitors. To date, preclinical studies have found that nearly 100 different types of small-molecule compounds can selectively/non-selectively inhibit or partially inhibit FLT3 protein kinase activity and the in vitro proliferation of cells as well as the in vivo xenograft tumor growth with FLT3 mutant expression of positive leukemia cell lines or leukemia patients. Some of these compounds have entered different stages of clinical trials, such as CEP701, CHIR-258, PKC412, MLN-518, sunitinib, AC220, XL-999, Sorafenib, Ponatinib, Crenolanib, ASP 2215, AKN-028, TAK-659, E6201, cabozantinib, PLX 3387, and FLX 925 (Smith B D et al., Single-agent CEP-701, a novel FLT3 inhibitor, shows biologic and clinical activity in patients with relapsed or refractory acute myeloid leukemia. Blood. 2004; 103 (10):3669-76; Lopes de Menezes D E et al., CHIR-258: a potent inhibitor of FLT3 kinase in experimental tumor xenograft models of human acute myelogenous leukemia. Clin Cancer Res. 2005; 11(14):5281-91; Weisberg E et al., Inhibition of mutant FLT3 receptors in leukemia cells by the small molecule tyrosine kinase inhibitor PKC412. Cancer Cell. 2002; 1 (5):433-43; Zarrinkar P P et al., AC220 is a uniquely potent and selective inhibitor of FLT3 for the treatment of acute myeloid leukemia (AML). Blood. 2009; 114(14):2984-92; Kelly L M et al., CT53518, a novel selective FLT3 antagonist for the treatment of acute myelogenous leukemia (AML). Cancer Cell. 2002; 1(5):421-32; Griswold I J et al., Effects of MLN518, a dual FLT3 and KIT inhibitor, on normal and malignant hematopoiesis. Blood. 2004; 104(9):2912-8; Smith C C et al., Crenolanib is a selective type I pan-FLT3 inhibitor. Proc Natl Acad Sci USA. 2014; 111(14):5319-24; Zimmerman El et al., Crenolanib is active against models of drug-resistant FLT3-ITD-positive acute myeloid leukemia. Blood. 2013; 122 (22):3607-15; Safaian N N et al., Sorafenib (Nexavar) induces molecular remission and regression of extramedullary disease in a patient with FLT3-ITD+ acute myeloid leukemia. Leuk Res. 2009; 33 (2):348-50; Zhang W et al., Mutant FLT3: a direct target of sorafenib in acute myelogenous leukemia. J Natl Cancer Inst. 2008; 100(3):184-98).
In spite that some FLT3 inhibitors showing encouraging results in the early stages of clinical trials with the condition of AML patients showing improvement, most compounds did not show the expected clinical effects in the later stages of clinical trials when used as a monotherapy or as a combination treatment for AML patients, and currently, no single FLT3 selective inhibitor has been approved for the clinical treatment of AML anywhere in the world.
For the specificity of the AML disease, by summarizing and analyzing the preclinical and clinical trial data for existing FLT3 inhibitors, we found that most FLT3 inhibitors experience a variety of issues which limit their clinical effects. These problems include: (1) severe side effects; (2) affecting the growth and development of normal blood cells and reducing patient immunity; (3) acquiring drug resistance; (4) tumor lysis syndrome; (5) low patient response rate and (6) easy to relapse. Such issues are mainly related to the selectivity, activity, in vivo metabolism, toxicity and effectiveness of the compounds. (Kadia T M et al., New Drugs in Acute Myeloid Leukemia (AML). Ann Oncol. 2016; 27 (5): 77-8. Stein E M et al., Emerging therapeutic drugs for AML. Blood. 2016; 127 (1):71-8. Stein E M, Molecularly targeted therapies for acute myeloid leukemia. Hematology Am Soc Hematol Educ Program. 2015; (1):579-83).
At present, three human leukemia cell lines MV4-11, MOLM-13 and MOLM-14 are widely used for studies of FLT3-ITD expression positive cells in the world. The MV4-11 cells contain homozygous mutation (+/+) of FLT3 ITD and are attributed to human acute lymphoblastic mononuclear cell leukemia. MOLM-13 and MOLM-14 are sister cell lines from the same patient and contain FLT3 ITD/WT heterozygous mutation (+/−), which attributed to human acute myeloid leukemia (Quentmeier H et al., FLT3 mutations in acute myeloid leukemia cell lines. Leukemia. 2003; 17(1): 120-124). Many studies have demonstrated that the targeted inhibition of FLT3-ITD can effectively inhibit the growth of these three kinds of leukemia cells in vivo and in vitro. These cell lines, particularly MV4-11, have become common cellular models for the screening and identification of FLT3-ITD selective inhibitors.
In the present invention, by using (1) FLT3-ITD expression positive cell lines MV4-11 and MOLM-13, (2) FLT3 gene wild-type positive cell line RS4 11 with high expression, (3) other clinically common oncogene-expressing positive leukemia cell lines as well as different types of solid tumor cell lines as the cell models, the inventor(s) are dedicated to developing a new type of compound with high activity, high selectivity, good pharmacological effects and pharmacokinetics properties, and low side effects, which can be used to treat and/or prevent cancer, especially leukemia, as an effectively selective inhibitor of FLT3 tyrosine protein kinase (particularly FLT3 activating mutation).